Fig 1.
Induction of Glomerular E2A expression in Thy-1 nephritis rats.
Representative light microphotograph from sections of the Thy-1 nephritis rat glomeruli stained with periodic acid-Schiff (PAS) stain and immunofluorescence analyses of E2A protein expression in Thy-1 glomerulonephritis (A-F) and sham-operated (Sham) control rats (G–I) at day 0 (A, D, and, G), at day 4 (B, E, and H) and at day 12 (C, F, and, I) (n = 6 in each group). Original magnification for all panels was ×400.
Fig 2.
Association of cell proliferation and effect of siRNA-mediated E12 silencing on MCs.
(A) Ablation of E12 expression following siRNA knockdown was confirmed by Western blot. One of three independent experiments is shown. (B) Effects of RNAi-mediated silencing of E2A on cell proliferation of MCs for the indicated times. The siRNA specific for E2A or the control siRNA (siScramble) was transfected into MCs. Results represent as arbitrary units and are shown as mean values ± SDs of at least five independent experiments. NS, not significant.
Fig 3.
Physical interactions between E2A and FLASH.
(A) Interaction between E12-bHLH and FLASH in a yeast two-hybrid assay. pGBKT7-E12-bHLH with pGADT7-Id3 (positive control) or pGADT7-FLASH-truncated (ΔFLASH) were transformed into AH109 as the manufacturer’s instructions. Colonies were counted and calculated as colony-forming units (cfus). (B) CHO-K1 cells were transfected with Myc-tagged E12-bHLH and HA-tagged Id3 or ΔFLASH. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted (IB) with anti-Myc antibody (upper panel). Total cell lysates were blotted with anti-Myc antibody (middle panel) or anti-HA antibody (lower panel). One of three independent experiments is shown in each panel. (C) Co-immunoprecipitation of full-length FLASH and E12-bHLH E47-bHLH, full-length E12, or full-length E47 from co-transfected MC lysate was detected by immunoblot analysis. One of three independent experiments is shown in each panel. (D) Endogenous FLASH and E2A co-immunoprecipitation. Kidney lysates from Thy-1 glomerulonephritis or sham-operated (sham) rats at day 4 and day 12 were immunoprecipitated (IP). The presence of E2A and FLASH in the immunoprecipitates was determined by immunoblot (IB) with anti-E2A and anti-FLASH antibodies, respectively. (n = 3 in each group)
Fig 4.
Inhibitory effect of siRNA-mediated FLASH silencing on MC proliferation.
(A) Ablation of E12 and FLASH expression following siRNA knockdown was confirmed by Western blot. (B) Effects of RNAi-mediated silencing of E2A and FLASH on cell proliferation of MCs for the indicated times. The control siRNA (scrambled) or the siRNA specific for E2A alone, FLASH alone, and both E2A and FLASH was transfected into MCs. Data represent mean values ± SDs of at least five independent experiments. *P<0.01.
Fig 5.
FLASH silencing with siRNAs affects p21 expression in MCs.
Effects of RNAi-mediated silencing of FLASH in cultured mouse MCs. Scrambled siRNA (Scramble) was used as a control. (A) Equal amounts of cell lysates and total RNA were subjected to Western blot (A) and quantative RT-PCR (C), respectively. Since the MCs did not express p16 protein under the usual culture conditions, we have shown tha p16 expression data from p16-expressing MCs induced by cellular responses across multiple passage numbers. PC, positive control (late-passage mouse MCs). One of three independent experiments is shown in (A). (B) FLASH, p21, p27 and p53 protein levels were quantified by densitometric analysis and are expressed as the ratio between these proteins bands optical density and β-actin bands optical density. The quantitative comparison between control siRNA (scrambled) and the most effective siRNA specific for FLASH (RNAi-FLASH3 as KD) were analyzed. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001. (C) Real-time RT-PCR showing relative mRNA levels for FLASH, p16, p21, p27 and p53. The quantitative comparison between scrambled siRNA (Control) and the most effective siRNA specific for FLASH (RNAi-FLASH3 as KD) were analyzed. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001.
Fig 6.
E12-induced p21 expression was attenuated by FLASH in MCs.
(A) Western blot analyses of MCs transfected with full-length E12 and E47 plasmids, and empty vector (Mock). β-actin was used as an internal control. One of three independent experiments is shown. E12 and p21 protein levels were quantified by densitometric analysis and are expressed as the ratio between these proteins bands optical density and β-actin bands optical density. The quantitative comparison between the transfection of Empty vector (Empty) and the tranfection E12 expression vector (E12) were analyzed. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. *P<0.001. (B) Representative fluorescence staining pictures are shown. The induction of p21 in Thy-1 glomerulonephritis rats was followed by FLASH induction. The peaks of p21 and FLASH were observed at day 4 and day 12, respectively. FLASH expression in the glomeruli corresponded to the nuclei (DAPI in blue), and was shown in the merged image (Merge). Nuclei are illustrated by DAPI staining (blue). (C) Western blot confirmation of the attenuation of E12-induced p21 protein expression by the forced expression of FLASH. β-actin was used as an internal control. One of three independent experiments is shown. (D) FLASH silencing by specific siRNA clearly increased the protein level of p21 in MCs. β-actin was used as a loading control. One of three independent experiments is shown. (E) MCs were co-transfected in parallel with full-length E12 and increasing amounts of the FLASH expression plasmids (0.5, 1.5 and 3 μg), and transfected with FLASH expression plasmids alone (3 μg). For all transfection, the final concentration of DNA was adjusted to 5 μg using empty expression vector. Proteins were extracted and analyzed by Western blot analyses. β-actin was used as an internal control. One of three independent experiments is shown. (F) Silencing of p53 by specific siRNA clearly increased the protein level of p53 independetly of E12-induced p21expression in MCs. β-actin was used as a loading control. One of three independent experiments is shown.
Fig 7.
Effects of E12, p21 and FLASH on cellular senescence in MCs.
(A) SA-β-gal staining at 48 h after transfection of expression plasmids for E12, p21 and FLASH, or empty vector (Mock) in MCs with 24 h-treatment with or without BeSO4 shows induction of senescence. Arrows show cells with morphological changes. (B) Cellular senescence by measuring SA-β-Gal activity using a fluorometric substrate. MCs with treatments as shown in (A) were lysed. Lysates were allowed to incubate with SA-β-Gal substrate for 1 hr at 37°C. Background-subtracted relative fluorescence units (RFUs) were normalized to optical density; excited at 360 nm, and showed emission at 465 nm. Experiments were conducted in triplicate (error bars are ± SDs). *P<0.001, NS, not significant. (C) Cell number was counted using a hemocytometer at the indicated conditions. Quantitative data for cell numbers from five independent experiments are presented as means ± S.D. *P<0.05. (D) Cells were counted on the basis of their morphological changes and expressed as the percentage of the total cell population. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001. (E) SA-β-gal staining at 48 h after knockdown of E12, p21 and FLASH in MCs shows induction of senescence. Arrows show cells with morphological changes. (F) Cellular senescence by measuring SA-β-Gal activity. MCs with treatments as shown in (E) were lysed. RFUs were normalized to optical density; excited at 360 nm, and showed emission at 465 nm. Experiments were conducted in triplicate (error bars are ± S.D.). *P<0.001, NS, not significant. (G) The number of cells was determined microscopically using a hemocytometer at the indicated conditions. Quantitative data for cell numbers from five independent experiments are presented as means ± S.D. *P<0.05. (H) Cells were counted on the basis of their morphological changes and expressed as the percentage of the total cell population. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001.
Fig 8.
Negative regulation of TNF-α-mediated p21 expression through upregulation of FLASH in MCs.
(A) Western blot analyses of MCs exposed to TNF-α at the indicated concentrations for 12 h. β-actin was used as an internal control. One of three independent experiments is shown. (B) Time course of p21, p53 and FLASH expression levels in MCs treated with low (0.1 ng/ml) or high-dose (10 ng/ml) TNF-α. In addition, the effects of comparative high concentration of TNF-α on p21 expression in a dose-dependent manner for 36 and 72 h stimulation were monitored by Western blot analyses. One of three independent experiments is shown in each panel. β-actin was used as an internal control. FLASH and p21 protein levels were quantified by densitometric analysis and are expressed as the ratio between these proteins bands optical density. Results represent as arbitrary units and are shown as mean values ± SDs. NS, not significant. *P<0.001. (C) Regression analysis comparing the changes in p21 levels with changes in FLASH expression levels under high concentration of TNF-α stimulation, the expression level of p21 negatively correlated with the expression level of FLASH (R = 0.922; P < 0.01). (D) MCs were treated with only transfection reagent (Mock) or transfected with scrambled or FLASH siRNA. After the siRNA transfection and successive 24 h incubation, MCs were treated with low concentrations of TNF-α (0 or 0.1 ng/ml) for 12 h, and treated with high concentrations of TNF-α(10 ng/ml) for 48h, respectively. After these stimulation of TNF-α, the cells were harvested and subjected to western blot. One of three independent experiments is shown. (E) Western blot analyses of MCs transfected with full-length FLASH plasmid, and empty vector (Mock) under low concentration of TNF-α. β-actin was used as an internal control. One of three independent experiments is shown. (F) MCs were mock-transfected (Mock) or transfected with scrambled or E12 siRNA. After the siRNA transfection and successive 24 h incubation, MCs were treated with 0 or 0.5 ng/ml of TNF-α. β-actin was used as an internal control. One of three independent experiments is shown.
Fig 9.
Effects of p21 and FLASH under TNF-α stimulation on cellular senescence in MCs.
(A) SA-β-gal staining at 48 h after knockdown of p21 and FLASH in MCs in the absence or presence of TNF-α shows induction of senescence. Representative data from one out of five independent experiments are shown. (B) The number of cells was determined microscopically using a hemocytometer at the indicated conditions. (C) Cells were counted on the basis of their morphological changes and expressed as the percentage of the total cell population. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. *P<0.001.